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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 19 December 2017 to 27 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch no. 99671, supplied by the sponsor
- Expiration date of the lot/batch: 9 Oct 2019
- Purity test date:10 october 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (20±5°C)
- Stability under test conditions: not applicable
- Solubility and stability of the test substance in the solvent/vehicle: not applicable , no vehicle was used
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no reaction

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no treatment, the test item was used neat
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Neat

In vitro test system

Test system:
artificial membrane barrier model
Source species:
human
Cell type:
other: human-derived epidermal keratinocytes
Cell source:
other: not applicable
Source strain:
other: not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM
- Tissue batch number(s): 25875
- Production date: not specified
- Shipping date: not specified
- Delivery date: 23 January 2018
- Date of initiation of testing: 22 January 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:for 3 minutes exposure : at room temperature ; for 1 hour exposure : 37± 1°C
- Temperature of post-treatment incubation (if applicable): 37±1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: DPBS washing, 20 times
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: stock solution : 5mg/mL ; test solution : 1mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos Reader 2010 Flexi
- Wavelength: 570 nm
- Filter:no filter was used
- Filter bandwidth: no filter was used
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.438 ± 0.104 (pass)
- Barrier function: 8.04 hours (pass)
- Morphology: pass
- Contamination: sterile (pass)
- Reproducibility:pass

NUMBER OF REPLICATE TISSUES: duplicates were used per condition

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : not specified
- Procedure used to prepare the killed tissues (if applicable): killed tissue was not used, the procedure was not described
- N. of replicates : not applicable
- Method of calculation used: not applicable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 independent sequences : pre-test and main test

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50µL
- Concentration (if solution): vehicle was not used

VEHICLE not applicable

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): neat

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8M in solution with demineralised water
Duration of treatment / exposure:
3 minutes and 1 hour
Duration of post-treatment incubation (if applicable):
not applicable
Number of replicates:
duplicates were used per condition

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test item, 3 minutes exposure period, mean tissue viability (%)
Value:
91.5
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test item, 1 hour exposure period, mean tissue viability (%)
Value:
93.5
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The positive control condition showed techical proficiency of the test item

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Valid
- Acceptance criteria met for positive control: Valid
- Acceptance criteria met for variability between replicate measurements: Valid

Any other information on results incl. tables

Table1% Tissue Viability

Test Item

Positive Control

Incubation

91.5%

18.9%

3 min

93.5%

9.9%

1 h

 

Table 2Historical Data

Parameter

Optical Density Negative

Control

Optical Density Negative

Control

% Tissue

viability
Positive

Control

% Tissue

viability
Positive

Control

Incubation Time

3 min.

1 h

3 min.

1 h

Mean

1.932

1.882

24.6%

11.4%

Standard
Deviation

0.271

0.221

6.7%

4%

Range

1.197 – 3.077

1.377 – 2.571

9.6 – 57.3%

4.1 – 24.2%

Study17110901G820

2.076

1.991

18.9%

9.9%

 

 

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Non-corrosive
Conclusions:
Under the experimental condition of the study, the Triisononylamine did not increase decrease of tissue viability when applied on EpidermTm. Hence, the test substance was considered as Not Corrosive according to CLP criteria.
Executive summary:

This GLP compliant study was performed in order to evaluate the skin corrosion potential of Triisononylamine to human skin EpiDermTM in an in vitro study according to OECD Guideline 431 method.

Two tissues of the human skin model EpiDermTM were treated with the test item Triisononylamine for 3 minutes and 1 hour, respectively. The test item was applied to each tissue and spread to match the tissue size. Demineralised water was used as negative control and 8 M KOH was used as positive control.

After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a blue formazan. Formazan production was evaluated by measuring the optical density (OD) of the resulting solution. After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals thus showing the quality of the tissues. The OD was 2.1 (3 minutes experiment) and 2.0 (1 hour experiment).

The positive control showed clear corrosive effects for both treatment intervals. The mean relative tissue viability value was reduced to 9.9% after 1 hour treatment.

After 3 minutes treatment with the test item, the mean value of relative tissue viability was reduced to 91.5 %. This value is above the threshold for corrosion potential (50%). After 1 hour treatment, the mean value of relative tissue viability was reduced to 93.5%. This value, too, is above the threshold for corrosion potential (15%).

Under the experimental condition of the study, the Triisononylamine did not increase decrease of tissue viability when applied on EpidermTm tissue. Hence, the test substance was considered as Not Corrosive.